Small molecule protein arginine methyltransferase 5 (prmt5) inhibitors and methods of treatment

ABSTRACT

Provided are compounds of formulas (I), (II), (III), and (IV), which effectively inhibit protein arginine methyltransferase 5 (PRMT5). Also provided are methods of using the compounds, including a method of treating cancer, a method of inhibiting the activity of PRMT5 in a cell, and a method of treating a disease associated with increased activity of PRMT5.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.16/344,644, which is a U.S. national counterpart application ofinternational application serial No. PCT/US2017/058572 filed Oct. 26,2017 which claims the benefit of U.S. Provisional Patent Application No.62/534,969, filed Jul. 20, 2017, and U.S. Provisional Patent ApplicationNo. 62/413,341, filed Oct. 26, 2016, the disclosures of which are eachincorporated in their entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with Government support under Grant NumberTR001108 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Posttranslational modifications (PTMs) regulate protein function ineukaryotes and have been shown to play a role in a variety of cellularfunctions. In the past decade, research has greatly advanced theunderstanding of the role of PTMs of various signaling molecules thatlead to the development of a variety of diseases, including cancers(Karve et al., J. Amino Acids, 2011; 2011: 207691). Methylation oflysine and arginine is one of the most critical PTMs seen in nature andis implicated in a number of cellular processes, such as DNA damage andrepair, gene transcription and translation, and protein subcellularlocalization and translocation. Arginine methylation is carried out by agroup of enzymes termed protein arginine methyltransferases. Amongstthis family, protein arginine methyltransferase 5 (PRMT5) has beenimplicated in the development of a wide range of diseases. For example,the expression of PRMT5 is upregulated in a variety of cancers (e.g.,liver cancer, pancreatic cancer, breast cancer, prostate cancer, andlung cancer, as well as lymphoma and melanoma), neurodegenerativedisorders, inflammatory diseases, metabolic disorders, cardiovasculardiseases, autoimmune disorders, and blood disorders.

PRMT5 is an activator of NF-κB via dimethylating arginine 30 of the p65subunit of NF-κB. NF-κB is a critical eukaryotic transcription factorwhose family consists of five members: RelA (p65), RelB, cRel, NF-κB1(p50 and its precursor p105), and NF-κB2 (p52 and its precursor p100)(Ghosh et al., Annu Rev Immunol, 1998; 16: 225-260). NF-κB signaling canbe classified into canonical and non-canonical pathways. The canonicalpathway has been well established as a key contributor to development ofboth pancreatic ductal adenocarcinoma (PDAC) (Prabhu et al., Oncotarget,2014; 5: 10969-10975; Liou et al., J Cell Biol, 2013; 202: 563-577) andcolorectal cancer (CRC) (Agarwal et al., Oncogene, 2005; 24(6): 1021-31;Yu et al., Int J Colorectal Dis, 2004; 19: 18-22). In this pathway,inhibitor of κB (IκBα) sequesters the p65:p50 heterodimer in an inactivestate in the cytoplasm. When a cell receives extracellular signals, suchas stress or pro-inflammatory cytokines, IκB kinase phosphorylates IκBα,which leads to the degradation of IκB and release of the p65:p50 complexand the activation of NF-κB target genes (Gilmore, Oncogene, 2006; 25:6680-6684). A number of these downstream NF-κB target genes have beenimplicated in a wide range of diseases including cancer,neurodegenerative disorders, inflammatory diseases, metabolic disorders,cardiovascular diseases, autoimmune disorders, and blood disorders.Increased NF-κB activation is shown to be associated with a poor diseaseprognosis, and linked to developing resistance against chemotherapy(Arora et al., J Biol Chem, 2013; 288: 21197-207; Lind et al., Surgery,2001; 130: 363-69).

Accordingly, there is a need to identify new compounds for the treatmentof PRMT5-associated diseases. This invention provides such compounds andassociated methods.

BRIEF SUMMARY OF THE INVENTION

The invention provides compounds of formula (I), formula (II), formula(III), and formula (IV)

in which R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R^(1A), R^(2A), R^(1B), R^(2B),R^(3B), R^(4B), R^(5B), R^(6B), R^(7B), R^(8B), R^(9B), R^(10B), X,X^(1A), X^(1B), m, and n are described herein. It has been discoveredthat compounds defined by formula (I), formula (II), formula (III), orformula (IV) are effective in inhibiting PRMT5, thereby making thecompounds effective in treating diseases associated with increasedexpression or activity of PRMT5 (e.g., cancer).

The invention further provides a method of treating a cancer in asubject comprising administering a pharmaceutically effective amount ofa compound of formula (I), formula (II), formula (III), or formula (IV),or a pharmaceutically acceptable salt thereof to the subject.

Also provided is a method of inhibiting the activity of PRMT5 in a cellcomprising administering a pharmaceutically effective amount of acompound of formula (I), formula (II), formula (III), or formula (IV),or a pharmaceutically acceptable salt thereof to the cell.

The invention also provides a method of treating a disease associatedwith increased expression or activity of PRMT5 in a subject comprisingadministering a pharmaceutically effective amount of a compound offormula (I), formula (II), formula (III), or formula (IV), or apharmaceutically acceptable salt thereof to the subject.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 is a schematic illustration of the AlphaLISA technique for theidentification of small-molecule PRMT5 inhibitors. Biotinylatedsubstrate (histone H4) is incubated with PRMT5 and methyl donor,S-adenosyl-1-methionine (SAM). PRMT5 symmetrically dimethylates thethird arginine (R3) on biotin-H4 to form biotin-H4R3me2. This product isrecognized by Acceptor beads specific for this methylation site. Donorbeads have a streptavidin tag and bind to biotin-H4. Interaction betweenthe Acceptor and Donor beads emits a chemiluminescent signal, which isdetected by an ENVISION™ Reader (PerkinElmer, Waltham, Mass.). Themethylation activity of PRMT5 is proportional to the intensity of thissignal.

FIGS. 2A-D depict the experimental results of studies to determine thePRMT5 inhibitory activity of a compound of formula (II), a compound offormula (III), and a compound of formula (IV). FIG. 2A is a calculationof IC₅₀ of compound (IIa) using AlphaLISA, which was found to be ˜7.5μM. FIG. 2B is a calculation of IC₅₀ of compound (IIIa) using AlphaLISA,which was found to be ˜1.5 μM. FIG. 2C is a calculation of IC₅₀ ofcompound (IVa) using AlphaLISA, which was found to be ˜16.5 μM. FIG. 2Dis a co-immunoprecipitation-Western blot, showing that treatment with 20μM compound (IIa) for 24 hours inhibited the methylation of PRMT5substrate, p65, in 293-WT-p65-Flag cell lines. Flag beads were used topull down WT-p65-Flag and samples were then probed with anti-symmetricdimethyl arginine motif Ab (sdme-RG Ab) to detect dimethylation levelsof the p65 subunit.

FIGS. 3A-B depict the results of lead optimization studies to identifyPRMT5 inhibitors. FIG. 3A is a table showing the calculation of IC₅₀ ofcompounds (Ia), (If), and (Ig) compared to the parent compound (IIa) inHT-29 colon cancer cells. FIG. 3B is a table showing the calculation ofIC₅₀ of compounds (Ia), (Ib), (If), and (Ig) compared to the parentcompound (IIa) in PANC1 pancreatic cancer cells.

FIGS. 4A-D depict the experimental results of studies to determine theexpression level of PRMT5 in multiple cancer types. FIG. 4A is a Westernblot showing that PRMT5 protein expression is higher in PDAC cell lines,AsPC1, MiaPaCa2 and PANC1, as compared to a control pancreatic cellline, HPNE. β-actin was used as a loading control. FIG. 4B isimmunohistochemical (IHC) staining demonstrating that PRMT5 proteinexpression is higher in PDAC tumor tissue as compared to the tissue fromnormal (i.e., non-cancerous) controls. FIG. 4C is a Western blot showingthat PRMT5 protein expression is higher in CRC cell lines, HT29, HCT116and DLD1, as compared to a control colon cell line, FHC. β-Actin wasused as a loading control. FIG. 4D is IHC staining showing that PRMT5protein expression was higher in the polyp stage and advanced stages ofCRC, including stages II, III, IV, and the metastatic stage, compared tonormal, non-cancerous colon tissue.

FIGS. 5A-G depict the experimental results of studies to determine theeffect of increased PRMT5 activity in cells. FIG. 5A is Western blot,confirming stable PRMT5 overexpression and shPRMT5 knockdown in PANC1(left panel) and HT29 (right panel) cell lines. β-Actin was used as aloading control. FIGS. 5B and 5C are a cell proliferation assay, showingthe effect of PRMT5 overexpression and shPRMT5 knockdown on cell growthin PANC1 (FIG. 5B) and HT29 (FIG. 5C) cell lines. Cell proliferation wassignificantly higher in the WT-PRMT5 cell lines, while shPRMT5 cellsshowed the opposite effect in both PANC1 and HT29 cell lines. FIGS. 5Dand 5E are an anchorage-independent assay, showing the effect of PRMT5overexpression and shPRMT5 knockdown on the colony size and colonynumber in PANC1 (FIG. 5D) and HT29 (FIG. 5E) cells.Anchorage-independent growth was significantly higher in the WT-PRMT5cell lines, while significantly reduced in the shPRMT5 cells. FIGS. 5Fand 5G are a cell migration assay, showing the effect of PRMT5overexpression and shPRMT5 knockdown on cell migration in PANC1 (FIG.5F) and HT29 (FIG. 5G) cell lines. The upper panels show representativepictures of the respective wells with 20× magnification. The lower paneldenotes the quantification for the change in migration between the celllines. Cell migration was significantly higher in the WT-PRMT5overexpression cells, while significantly reduced in the shPRMT5 cells.The data represent the means±SD for three independent experiments.*P<0.05 vs. Ctrl group.

FIGS. 6A-C depict the results of experiments to determine the inhibitoryeffect of compound (Ia) in multiple cancer types. FIG. 6A is an MTTassay in PDAC cells (PANC1), demonstrating that cell viability decreasedsignificantly in the presence of increasing concentrations of compound(Ia). FIG. 6B is an MTT assay in CRC cells (HT29), showing that cellviability decreased significantly in the presence of increasedconcentrations of compound (Ia). FIG. 6C is a table, summarizing theIC₅₀ values for compound (Ia) in PDAC and CRC cells, respectively.

FIGS. 7A-D depict the results of experiments to determine the in vivoeffect of compound (Ia). FIGS. 7A and 7B show that no significantchanges in body weight were observed over the course of treatment ineither PANC1 (FIG. 7A) or HT29 (FIG. 7B) model after treatment with 20mg/kg of compound (Ia). (*P<0.05, n=4). FIGS. 7C and 7D are a tumorefficacy study, in which PANC1 (FIG. 7C) or HT29 (FIG. 7D) cells weresubcutaneously implanted in NSG mice. Tumor volumes were measured andinhibition of tumor growth was observed upon treatment with 20 mg/kg ofcompound (Ia) intraperitoneally for 3×/week, as compared to the vehiclecontrol. (*P<0.05, n=4).

FIGS. 8A-H depict the results of experiments to determine the inhibitoryeffect of compound (IIa) in multiple cancer types. FIGS. 8A-C are an MTTassay in PDAC cells (PANC1, MiaPaCa2, and AsPC1), demonstrating thatcell viability decreased significantly in the presence of increasingconcentrations of compound (IIa). FIGS. 8D-F are an MTT assay in CRCcells (HT29, HCT116, and DLD1), showing that cell viability decreasedsignificantly in the presence of increased concentrations of compound(IIa). FIG. 8G is a table, summarizing the IC₅₀ values for compound(IIa) in PDAC and CRC cells, respectively. FIG. 8H is ananchorage-independent assay, showing that with increasing concentrationsof compound (IIa), there was a significant decrease in theanchorage-independent growth ability in both PANC1 as well as HT29cells.

FIGS. 9A-G depict the results of experiments to determine the inhibitoryeffect of a commercially available PRMT5 inhibitor. FIGS. 9A-C are anMTT assay, showing that the commercially available PRMT5 inhibitor,EPZ015666, has lower efficacy to decrease cell viability in PDAC cells(PANC1, MiaPaCa2 and AsPC1) than that of compound (IIa). FIGS. 9D-F arean MTT assay, showing that EPZ015666 has lower efficacy to decrease cellviability in CRC cells (HT29, HCT116, and DLD1) than that of compound(IIa). The data represent the means±SD for three independentexperiments. *P<0.05 vs. Ctrl group. FIG. 9G is a table summarizing theIC₅₀ values for EPZ015666 in PDAC and CRC cell lines, respectively.

FIGS. 10A-D depict the results of experiments to determine the in vivoeffect of compound (IIa). FIGS. 10A and 10B show that no significantchanges in body weight were observed over the course of treatment ineither PANC1 (FIG. 10A) or HT29 (FIG. 10B) model after treatment with 20mg/kg of compound (IIa). (*P<0.05, n=4). FIGS. 10C and 10D are a tumorefficacy study, in which PANC1 (FIG. 10C) or HT29 (FIG. 10D) cells weresubcutaneously implanted in NSG mice. Tumor volumes were measured andinhibition of tumor growth was observed upon treatment with 20 mg/kg ofcompound (IIa) intraperitoneally for 3×/week, as compared to the vehiclecontrol. (*P<0.05, n=4).

FIGS. 11A-L depicts the results of experiments to determine the effectof compound (IIa) on PRMT5 induced NF-κB activation. FIGS. 11A and 11Bare a NF-κB luciferase assay, showing that overexpression of WT-PRMT5led to NF-κB activation, while shPRMT5 resulted in the opposite effectin both PANC1 (FIG. 11A) and HT29 cells (FIG. 11B). FIGS. 11C and 11Dare a luciferase assay, showing a decrease in NF-κB activation withincreasing concentrations of compound (IIa) in PANC1 (FIG. 11C) and HT29cells (FIG. 11D), respectively. EPZ015666 needed a higher concentrationin order to reach a similar level of NF-κB inhibition as that ofcompound (IIa). The data represent the means±SD for three independentexperiments. *P<0.05 vs. Ctrl group. FIGS. 11E-H is a qPCR analysis,showing that overexpression of PRMT5 significantly enhancedIL-1β-triggered typical NF-κB target genes expression, TNFα and IL8,while shPRMT5 exhibited the opposite effect, in both PANC1 (FIGS. 11Eand 11F) and HT29 cells (FIGS. 11G and 11H). The data represent themeans±SD for three independent experiments. *P<0.05 vs. Ctrl group;#P<0.05 vs. Ctrl+IL-1β-treated group. FIGS. 11I-L are a qPCR analysis,showing that treatment with compound (IIa) dramatically decreased NF-κBtarget genes (TNFα and IL8) expression, in both PANC1 (FIGS. 11I and11J) and HT29 cells (FIGS. 11K and 11L). The data represent the means±SDfor three independent experiments. *P<0.5 vs. Ctrl group.

FIGS. 12A-M depict the results of experiments to determine theinhibitory effect of compound (IIIa) and compound (IVa) in multiplecancer types. FIGS. 12A-12C are MTT assays in PDAC cells (PANC1 (FIG.12A), MiaPaCa2 (FIG. 12B), and AsPC1 (FIG. 12C)), demonstrating thatcell viability decreased significantly in the presence of increasingconcentrations of compound (IIIa). FIGS. 12D-12F are MTT assays in CRCcells (HT29 (FIG. 12D), HTC116 (FIG. 12E), and DLD1 (FIG. 12F)),demonstrating that cell viability decreased significantly in thepresence of increasing concentrations of compound (IIIa). FIGS. 12G-12Iare MTT assays in PDAC cells (PANC1 (FIG. 12G), MiaPaCa2 (FIG. 12H), andAsPC1 (FIG. 12I)), demonstrating that cell viability decreasedsignificantly in the presence of increasing concentrations of compound(IVa). FIGS. 12J-12L are MTT assays in CRC cells (HT29 (FIG. 12J),HTC116 (FIG. 12K), and DLD1 (FIG. 12L)), demonstrating that cellviability decreased significantly in the presence of increasingconcentrations of compound (IVa). FIG. 12M is a table, summarizing theIC₅₀ values for compound (IIIa) and compound (IVa) in PDAC and CRCcells, respectively.

FIGS. 13A-B depict the results of experiments to identify the expressionof PRMT5 in neurodegenerative disorders. FIG. 13A is a Western blotshowing the expression of PRMT5 in cell lysate with PRMT5 overexpression(lane 2), non-transgenic B6 mice (6M) (lanes 3-5), hTau (humanized taumice) (3M) (lanes 6-7), hTau (6M) (lanes 8-9), htau;Trem2−/− mice (6M)(lanes 10-11), hTau (18M) (lanes 12-13), and APPPS1 amyloid mice (lanes14-15). FIG. 13B is a graph depicting the Western blot data of PRMT5expression. *P<0.5 vs. Ctrl group.

FIGS. 14A-B depicts the results of experiments to show the effect ofcompound (IIa) in neurological disorders. FIG. 14 shows the dopaminelevels (FIG. 14A) and serotonin levels (FIG. 14B) in the striatum ofrats treated with 7.5 mg/kg methamphetamine followed by 4 injections of1 mg/kg compound (IIa) given at 12 hour intervals beginning 12 hoursafter methamphetamine treatment. (n=5). *P<0.05 vs. Ctrl group.

FIG. 15 is a graph showing the expression level of PRMT5 in multiplehuman tissues, including heart tissue, as determined using the TiGERdatabase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compounds of formulas (I)-(IV) thathave been discovered to be effective in inhibiting PRMT5, which enablesthe effective use of the compounds in treating various diseasesassociated with an increased expression or activity of PRMT5.

In particular, the present invention provides a compound of formula (I)

wherein

R¹ is hydrogen, halo, C₁-C₈ alkyl, C₃-C₆ cycloalkyl, heterocycloalkyl,aryl, or heteroaryl;

R² is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, C₁-C₈alkoxy, C₃-C₆ cycloalkyloxy, aryloxy, halo, C₁-C₈ haloalkoxy, C₁-C₈haloalkyl, haloaryl, haloaryloxy, —CN, —NO₂, —(CH₂)_(n)C(O)R⁵,—(CH₂)_(n)CO₂R⁵, —(CH₂)_(n)C(O)NR⁵R⁶, —(CH₂)_(n)NR⁵C(O)R⁶, or—(CH₂)_(n)NR⁵R⁶;

R³ is hydroxy, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆cycloalkylalkyl, C₁-C₈ alkoxy, C₃-C₆ cycloalkyloxy, heterocycloalkyl,aryl, arylalkyl, heteroaryl, aryloxy, halo, C₁-C₈ haloalkyl, C₁-C₈haloalkoxy, haloaryl, haloaryloxy, —CN, —NO₂, —C(O)R⁵, —CO₂R⁵,—C(O)NR⁵R⁶, —NR⁵C(O)R⁶, —(CH₂)_(n)NR⁵R⁶, —(CH₂)_(n)SO₂NR⁵R⁶,—(CH₂)_(n)SO₂R⁵, aryl; or

two R³ moieties and the phenyl group to which they are attached form anaphthyl group that is optionally substituted;

R⁴ is H, hydroxy, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₁-C₈haloalkyl, —CN, —NO₂, —(CH₂)_(n)NR⁵R⁶, heterocycloalkyl, aryl, orheteroaryl;

X is a bond, —(CH₂)_(o)CR⁵R⁶—, —CR⁵R⁶(CH₂)_(o)—, —(CH₂)_(o)NR⁵—,—NR⁵(CH₂)_(o)—, —(CH₂)_(o)O—, or —O(CH₂)_(o)—,

R⁵ and R⁶ are the same or different and each is H or C₁-C₈ alkyl;

m, n, and o are the same or different and each is 0 or an integer from1-5, or

a pharmaceutically acceptable salt thereof.

In an embodiment of the compound of formula (I), R¹ is hydrogen, halo,C₁-C₈ alkyl, C₃-C₆ cycloalkyl, aryl, heterocycloalkyl selected from thegroup consisting of isoxazolyl, thiazolinyl, imidazolidinyl,piperazinyl, homopiperazinyl, pyrrolyl, pyrrolinyl, pyrazolyl, pyranyl,piperidyl, oxazolyl, and morpholinyl, or heteroaryl selected from thegroup consisting of pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl,benzimidazolyl, triazinyl, imidazolyl, (1,2,3)-triazolyl,(1,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, pyrrolyl, thienyl,isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl. Preferably, R¹ ishalo, heterocycloalkyl selected from the group consisting of isoxazolyl,thiazolinyl, imidazolidinyl, piperazinyl, homopiperazinyl, pyrrolyl,pyrrolinyl, pyrazolyl, pyranyl, piperidyl, oxazolyl, and morpholinyl, orheteroaryl selected from the group consisting of pyridinyl, pyridazinyl,pyrimidyl, pyrazinyl, benzimidazolyl, triazinyl, imidazolyl,(1,2,3)-triazolyl, (1,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl,pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl.

In any of the foregoing embodiments of the compound of formula (I), R²is halo, C₁-C₈ haloalkoxy, C₁-C₈ haloalkyl, haloaryl, or haloaryloxy.

In any of the foregoing embodiments of the compound of formula (I), R³is C₁-C₈ alkyl, halo, or C₁-C₈ haloalkyl.

In any of the foregoing embodiments of the compound of formula (I), R⁴is H.

In any of the foregoing embodiments of the compound of formula (I), X isa bond, —(CH₂)_(o)NR⁵—, or —NR⁵(CH₂)_(o)—. Preferably, X is a bond or—NH(CH₂)_(o)— (e.g., —NH(CH₂)— or —NH(CH₂)₂—).

In any of the foregoing embodiments of the compound of formula (I), m is1.

In any of the foregoing embodiments of the compound of formula (I), n is0, 1, or 2.

In any of the foregoing embodiments of the compound of formula (I), o is1 or 2.

In any of the foregoing embodiments of R⁵ and R⁶ are each H.

In any of the foregoing embodiments of the compound of formula (I), R¹is heterocycloalkyl selected from the group consisting of isoxazolyl,thiazolinyl, imidazolidinyl, piperazinyl, homopiperazinyl, pyrrolyl,pyrrolinyl, pyrazolyl, pyranyl, piperidyl, oxazolyl, and morpholinyl, orheteroaryl selected from the group consisting of pyridinyl, pyridazinyl,pyrimidyl, pyrazinyl, benzimidazolyl, triazinyl, imidazolyl,(1,2,3)-triazolyl, (1,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl,pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl.

In an embodiment of the compound of formula (I), R′ is halo,heterocycloalkyl selected from the group consisting of isoxazolyl,thiazolinyl, imidazolidinyl, piperazinyl, homopiperazinyl, pyrrolyl,pyrrolinyl, pyrazolyl, pyranyl, piperidyl, oxazolyl, and morpholinyl, orheteroaryl selected from the group consisting of pyridinyl, pyridazinyl,pyrimidyl, pyrazinyl, benzimidazolyl, triazinyl, imidazolyl,(1,2,3)-triazolyl, (1,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl,pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl;R² is halo; R³ is C₁-C₈ alkyl or halo; R⁴ is H; X is a bond or—NR⁵(CH₂)_(o)—, R⁵ is H; m is 1; and o is 1 or 2.

In an aspect of the invention, R¹ is heterocycloalkyl selected from thegroup consisting of isoxazolyl, thiazolinyl, imidazolidinyl,piperazinyl, homopiperazinyl, pyrrolyl, pyrrolinyl, pyrazolyl, pyranyl,piperidyl, oxazolyl, and morpholinyl, or heteroaryl selected from thegroup consisting of pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl,benzimidazolyl, triazinyl, imidazolyl, (1,2,3)-triazolyl,(1,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, pyrrolyl, thienyl,isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl; R² is halo; R³ isC₁-C₈ alkyl or halo; R⁴ is H; X is a bond or —NR⁵(CH₂)_(o)—, R⁵ is H; mis 1; and o is 1 or 2.

In any of the foregoing embodiments, the compound of formula (I) is inthe form of a pharmaceutically acceptable salt.

Exemplary compounds of formula (I) include compounds (Ia), (Ib), (Ic),(Id), (Ie), (If), and (Ig), are set forth below. Pharmaceuticallyacceptable salts of these exemplary compounds are also envisioned.

The present invention also provides a compound of formula (II):

wherein

R⁵ is halo, heterocycloalkyl, or heteroaryl;

R⁶ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, C₁-C₈alkoxy, C₃-C₆ cycloalkyloxy, aryloxy, halo, C₁-C₈ haloalkoxy, C₁-C₈haloalkyl, haloaryl,

haloaryloxy, —CN, —NO₂, —(CH₂)_(n)C(O)R⁹, —(CH₂)_(n)CO₂R⁹,—(CH₂)_(n)C(O)NR⁹R¹⁰, —(CH₂)_(n)NR⁹C(O)R¹⁰, —(CH₂)_(n)NR⁹R¹⁰,—NR¹¹(CH₂)_(n)NR⁹R¹⁰; or

two R⁶ moieties and the phenyl group to which they are attached form anaphthyl group that is optionally substituted;

R⁷ is hydroxy, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆cycloalkylalkyl, C₁-C₈ alkoxy, C₃-C₆ cycloalkyloxy, heterocycloalkyl,aryl, arylalkyl, heteroaryl, aryloxy, halo, C₁-C₈ haloalkyl, C₁-C₈haloalkoxy, haloaryl,

haloaryloxy, —CN, —NO₂, —C(O)R⁹, —CO₂R⁹, —C(O)NR⁹R¹⁰, —NR⁹C(O)R¹⁰,—(CH₂)_(n)NR⁹R¹⁰, —(CH₂)_(n)SO₂NR⁹R¹⁰, —(CH₂)_(n)SO₂R⁹, aryl; or

two R⁷ moieties and the phenyl group to which they are attached form anaphthyl group that is optionally substituted;

R⁸ is H, hydroxy, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₁-C₈haloalkyl, —CN, —NO₂, —(CH₂)_(n)NR⁹R¹⁰, heterocycloalkyl, aryl, orheteroaryl;

X is a bond, —(CH₂)_(o)CR⁹R¹⁰—, —CR⁹R¹⁰(CH₂)_(o)—, —(CH₂)_(o)NR⁹—,—NR⁹(CH₂)_(o)—, —(CH₂)_(o)O—, or —O(CH₂)_(o)—,

R⁹, R¹⁰, and R¹¹ are the same or different and each is H or C₁-C₈ alkyl;

m, n, and o are the same or different and each is 0 or an integer from1-5, or

a pharmaceutically acceptable salt thereof.

In an embodiment of the compound of formula (II), R¹ is hydrogen, halo,C₁-C₈ alkyl, C₃-C₆ cycloalkyl, aryl, heterocycloalkyl selected from thegroup consisting of isoxazolyl, thiazolinyl, imidazolidinyl,piperazinyl, homopiperazinyl, pyrrolyl, pyrrolinyl, pyrazolyl, pyranyl,piperidyl, oxazolyl, and morpholinyl, or heteroaryl selected from thegroup consisting of pyridinyl, pyridazinyl, pyrimidyl, pyrazinyl,benzimidazolyl, triazinyl, imidazolyl, (1,2,3)-triazolyl,(1,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, pyrrolyl, thienyl,isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl. Preferably, R¹ ishalo, heterocycloalkyl selected from the group consisting of isoxazolyl,thiazolinyl, imidazolidinyl, piperazinyl, homopiperazinyl, pyrrolyl,pyrrolinyl, pyrazolyl, pyranyl, piperidyl, oxazolyl, and morpholinyl, orheteroaryl selected from the group consisting of pyridinyl, pyridazinyl,pyrimidyl, pyrazinyl, benzimidazolyl, triazinyl, imidazolyl,(1,2,3)-triazolyl, (1,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl,pyrrolyl, thienyl, isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl.In another embodiment of the compound of formula (II), R¹ is substitutedphenyl, as described herein, including phenyl substituted with C₁-C₈alkyl, halo, or hydroxy. In a specific example, R¹ is 3,4-di-C₁-C₈alkylphenyl (e.g., 3,4-dimethylphenyl).

In any of the foregoing embodiments of the compound of formula (II), R²is halo, C₁-C₈ haloalkoxy, C₁-C₈ haloalkyl, haloaryl, or haloaryloxy.Alternatively, R² is —NR⁷(CH₂)_(n)NR⁵R⁶. In an aspect of thisembodiment, R⁵ and R⁶ are both C₁-C₈ alkyl, R⁷ is H, and/or n is 1 or 2.Preferably, R² is —NR⁷(CH₂)_(n)NR⁵R⁶; R⁵ and R⁶ are methyl; R⁷ is H; andn is 2.

In any of the foregoing embodiments of the compound of formula (II), R³is C₁-C₈ alkyl, halo, or C₁-C₈ haloalkyl.

In any of the foregoing embodiments of the compound of formula (II), R⁴is H.

In any of the foregoing embodiments of the compound of formula (II), Xis a bond, —(CH₂)_(o)NR⁵—, or —NR⁵(CH₂)_(o)—. Preferably, X is a bond or—NH(CH₂)_(o)— (e.g., —NH—, —NH(CH₂)— or —NH(CH₂)₂—).

In any of the foregoing embodiments of the compound of formula (II), mis 0 or 1. In some preferred embodiments, m is 0, and the phenyl ringdoes not include any substituents.

In any of the foregoing embodiments of the compound of formula (I), n is0, 1, or 2.

In any of the foregoing embodiments of the compound of formula (II), ois 0, 1, or 2.

In any of the foregoing embodiments of the compound of formula (II) R⁵and R⁶ are each H.

An exemplary compound of formula (II) is compound (IIa).Pharmaceutically acceptable salts of the exemplary compound are alsoenvisioned.

In any of the foregoing embodiments of the compound of formula (I) orformula (II), IV preferably is not chloro. In a preferred embodiment,the compound of formula (I) or (II) is not4,6-dichloro-1-(p-tolyl)-1H-pyrazolo[3,4-d]pyrimidine or4,6-dichloro-1-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidine.

The present invention also provides a compound of formula (III):

wherein

R^(1A) and R^(2A) are the same or different and each is hydrogen, C₁-C₈alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkylalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, hydroxy, C₁-C₈ alkoxy,C₃-C₆ cycloalkyloxy, aryloxy, halo, C₁-C₈ haloalkoxy, C₁-C₈

haloalkyl, haloaryl,haloaryloxy, —CN, —NO₂, —(CH₂)_(n)C(O)R^(11A), —(CH₂)_(n)CO₂R^(11A),—(CH₂)_(n)C(O)NR^(11A)R^(12A), —(CH₂)_(n)NR^(11A)C(O)R^(12A), or—(CH₂)_(n)NR^(11A)R^(12A); wherein each moiety other than hydrogen,hydroxy, halo, —CN, and —NO₂ is optionally substituted;

X^(1A) is O, S, or —NR^(11A);

R^(11A) and R^(12A) are the same or different and each is H or C₁-C₈alkyl;

m is an integer from 1-4; and

n is 0 or an integer from 1-5, or

a pharmaceutically acceptable salt thereof.

An exemplary compound of formula (III) is compound (IIIa).Pharmaceutically acceptable salts of the exemplary compound also areenvisioned.

The present invention further provides a compound of formula (IV):

wherein

R^(1B), R^(2B), R^(3B), R^(4B), R^(5B), R^(6B), R^(7B), R^(8B), R^(9B),and R^(10B) are the same or different and each is hydrogen, C₁-C₈ alkyl,C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkylalkyl,heterocycloalkyl, aryl, arylalkyl, heteroaryl, hydroxy, C₁-C₈ alkoxy,C₃-C₆ cycloalkyloxy, aryloxy, halo, C₁-C₈ haloalkoxy, C₁-C₈ haloalkyl,haloaryl,

haloaryloxy, —CN, —NO₂, —(CH₂)_(n)C(O)R^(11A), —(CH₂)_(n)CO₂R^(11A),—(CH₂)_(n)C(O)NR^(11A)R^(12A), —(CH₂)_(n)NR^(11A)C(O)R^(12A), or—(CH₂)_(n)NR^(11A)R^(12A); wherein each moiety other than hydrogen,hydroxy, halo, —CN, and —NO₂ is optionally substituted;

X^(1B) is O, S, or —NR^(11B); and

R^(11B) and R^(12B) are the same or different and each is H or C₁-C₈alkyl, or

a pharmaceutically acceptable salt thereof.

An exemplary compound of formula (IV) is compound (IVa).Pharmaceutically acceptable salts of the exemplary compound are alsoenvisioned.

In any of the embodiments above, the term “alkyl” implies astraight-chain or branched alkyl substituent containing from, forexample, from about 1 to about 8 carbon atoms, e.g., from about 1 toabout 6 carbon atoms. Examples of alkyl group include methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,isopentyl, n-hexyl, and the like. This definition also applies wherever“alkyl” occurs as part of a group, such as, e.g., in C₃-C₆cycloalkylalkyl, hydroxyalkyl, haloalkyl (e.g., monohaloalkyl,dihaloalkyl, and trihaloalkyl), cyanoalkyl, aminoalkyl, alkylamino,dialkylamino, arylalkyl, etc. The alkyl can be substituted orunsubstituted, as described herein. Even in instances in which the alkylis an alkylene chain (e.g., —(CH₂)_(n)—), the alkyl group can besubstituted or unsubstituted. An example of a substituted alkylene chainincludes —CF₂-cyclopropyl.

In any of the embodiments above, the term “alkenyl,” as used herein,means a linear alkenyl substituent containing from, for example, about 2to about 8 carbon atoms (branched alkenyls are about 3 to about 8carbons atoms), e.g., from about 3 to about 6 carbon atoms (branchedalkenyls are about 3 to about 6 carbons atoms). In accordance with anembodiment, the alkenyl group is a C₂-C₄ alkenyl. Examples of alkenylgroup include ethenyl, allyl, 2-propenyl, 1-butenyl, 2-butenyl,1-pentenyl, 2-pentenyl, 3-pentenyl, 1-hexenyl, and the like. The alkenylcan be substituted or unsubstituted, as described herein.

In any of the embodiments above, the term “cycloalkyl,” as used herein,means a cyclic alkyl moiety containing from, for example, 3 to 6 carbonatoms or from 5 to 6 carbon atoms. Examples of such moieties includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. Thecycloalkyl can be substituted or unsubstituted, as described herein. Forexample, a substituted cycloalkyl includes a halo- orhaloalkyl-substituted cyclopropyl, such as 2-fluorocyclopropyl,2,2-difluorocyclopropyl, 1-(trifluoromethyl)cyclopropyl, and2-(trifluoromethyl)cyclopropyl.

In any of the embodiments above, the term “hydroxy” refers to the group—OH.

In any of the embodiments above, the terms “alkoxy” and “cycloalkyloxy”embrace linear or branched alkyl and cycloalkyl groups, respectively,that are attached to a divalent oxygen. The alkyl and cycloalkyl groupsare the same as described herein. The term “aryloxy” refers tosubstituents that have an aryl group attached to divalent oxygen. Thearyl group is the same as described herein.

In any of the embodiments above, the term “halo” refers to a halogenselected from fluorine, chlorine, bromine, and iodine.

In any of the embodiments above, the term “aryl” refers to a mono, bi,or tricyclic carbocyclic ring system having one, two, or three aromaticrings, for example, phenyl, naphthyl, anthracenyl, or biphenyl. The term“aryl” refers to an unsubstituted or substituted aromatic carbocyclicmoiety, as commonly understood in the art, and includes monocyclic andpolycyclic aromatics such as, for example, phenyl, biphenyl, naphthyl,anthracenyl, pyrenyl, and the like. An aryl moiety generally containsfrom, for example, 6 to 30 carbon atoms, from 6 to 18 carbon atoms, from6 to 14 carbon atoms, or from 6 to 10 carbon atoms. It is understoodthat the term aryl includes carbocyclic moieties that are planar andcomprise 4n+2 π electrons, according to Hückel's Rule, wherein n=1, 2,or 3. This definition also applies wherever “aryl” occurs as part of agroup, such as, e.g., in haloaryl (e.g., monohaloaryl, dihaloaryl, andtrihaloaryl), arylalkyl, etc. The aryl can be substituted orunsubstituted, as described herein.

In any of the embodiments above, the term “heteroaryl” refers toaromatic 5 or 6 membered monocyclic groups, 9 or 10 membered bicyclicgroups, and 11 to 14 membered tricyclic groups which have at least oneheteroatom (O, S, or N) in at least one of the rings. Each ring of theheteroaryl group containing a heteroatom can contain one or two oxygenor sulfur atoms and/or from one to four nitrogen atoms provided that thetotal number of heteroatoms in each ring is four or less and each ringhas at least one carbon atom. The fused rings completing the bicyclicand tricyclic groups may contain only carbon atoms and may be saturated,partially saturated, or unsaturated. The nitrogen and sulfur atoms mayoptionally be oxidized, and the nitrogen atoms may optionally bequaternized. Heteroaryl groups which are bicyclic or tricyclic mustinclude at least one fully aromatic ring but the other fused ring orrings may be aromatic or non-aromatic. The heteroaryl group may beattached at any available nitrogen or carbon atom of any ring.Illustrative examples of heteroaryl groups are pyridinyl, pyridazinyl,pyrimidyl, pyrazinyl, benzimidazolyl, triazinyl, imidazolyl, (1,2,3)-and (1,2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, pyrrolyl, thienyl,isothiazolyl, thiazolyl, isoxazolyl, and oxadiazolyl. The heteroaryl canbe substituted or unsubstituted, as described herein.

The term “heterocycloalkyl” means a stable, saturated, or partiallyunsaturated monocyclic, bicyclic, and spiro ring system containing 3 to7 ring members of carbon atoms and other atoms selected from nitrogen,sulfur, and/or oxygen. In an aspect, a heterocycloalkyl is a 5, 6, or7-membered monocyclic ring and contains one, two, or three heteroatomsselected from nitrogen, oxygen, and sulfur. The heterocycloalkyl may beattached to the parent structure through a carbon atom or through anyheteroatom of the heterocycloalkyl that results in a stable structure.Examples of such heterocycloalkyl rings are isoxazolyl, thiazolinyl,imidazolidinyl, piperazinyl, homopiperazinyl, pyrrolyl, pyrrolinyl,pyrazolyl, pyranyl, piperidyl, oxazolyl, and morpholinyl. Theheterocycloalkyl can be substituted or unsubstituted, as describedherein.

In any of the embodiments above, the alkyl, alkoxy, and alkylaminogroups can be linear or branched.

In other aspects, any substituent that is not hydrogen (e.g., C₁-C₈alkyl, C₂-C₈ alkenyl, C₂-C₈ alkynyl, C₃-C₆ cycloalkyl, C₃-C₆cycloalkylalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, orheterocycloalkyl) can be an optionally substituted moiety. Thesubstituted moiety typically comprises at least one substituent (e.g.,1, 2, 3, 4, 5, 6, etc.) in any suitable position (e.g., 1-, 2-, 3-, 4-,5-, or 6-position, etc.). When an aryl group is substituted with asubstituent, e.g., halo, amino, alkyl, OH, alkoxy, and others, thearomatic ring hydrogen is replaced with the substituent and this cantake place in any of the available hydrogens, e.g., 2, 3, 4, 5, and/or6-position wherein the 1-position is the point of attachment of the arylgroup in the compound of the present invention. Suitable substituentsinclude, e.g., halo, alkyl, alkenyl, alkynyl, hydroxy, nitro, cyano,amino, alkylamino, alkoxy, aryloxy, aralkoxy, carboxyl, carboxyalkyl,carboxyalkyloxy, amido, alkylamido, haloalkylamido, aryl, heteroaryl,and heterocycloalkyl, each of which is described herein. In someinstances, the substituent is at least one alkyl, halo, and/or haloalkyl(e.g., 1 or 2).

In any of the embodiments above, whenever a range of the number of atomsin a structure is indicated (e.g., a C₁₋₁₂, C₁₋₈, C₁₋₆, or C₁₋₄ alkyl,cycloalkyl, etc.), it is specifically contemplated that any sub-range orindividual number of carbon atoms falling within the indicated rangealso can be used. Thus, for instance, the recitation of a range of 1-8carbon atoms (e.g., C₁-C₈), 1-6 carbon atoms (e.g., C₁-C₆), 1-4 carbonatoms (e.g., C₁-C₄), 1-3 carbon atoms (e.g., C₁-C₃), or 2-8 carbon atoms(e.g., C₂-C₈) as used with respect to any chemical group (e.g., alkyl,cycloalkyl, etc.) referenced herein encompasses and specificallydescribes 1, 2, 3, 4, 5, 6, 7, and/or 8 carbon atoms, as appropriate, aswell as any sub-range thereof (e.g., 1-2 carbon atoms, 1-3 carbon atoms,1-4 carbon atoms, 1-5 carbon atoms, 1-6 carbon atoms, 1-7 carbon atoms,1-8 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5 carbon atoms,2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms, 3-4 carbon atoms,3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8 carbon atoms,4-5 carbon atoms, 4-6 carbon atoms, 4-7 carbon atoms, 4-8 carbon atoms,etc., as appropriate).

The subscript “m” represent the number of substituents of R³, in whicheach substituent of R³, can be the same or different. The subscripts mcan be either 0 or an integer from 1-5 (i.e., 1, 2, 3, 4, or 5). When mis 0, then the R³ is not present in the compound of formula (I) or (II).The subscripts “n” and “o” represent the number of methylene repeatunits. The subscripts n and o are either 0 or an integer from 1-5 (i.e.,1, 2, 3, 4, or 5). When n or o is 0, then the respective moiety does notcontain any methylene repeat units.

In any of the embodiments above, the phrase “salt” or “pharmaceuticallyacceptable salt” is intended to include nontoxic salts synthesized fromthe parent compound which contains a basic or acidic moiety byconventional chemical methods. Generally, such salts can be prepared byreacting the free acid or base forms of these compounds with astoichiometric amount of the appropriate base or acid in water or in anorganic solvent, or in a mixture of the two. For example, an inorganicacid (e.g., hydrochloric acid, sulfuric acid, phosphoric acid, orhydrobromic acid), an organic acid (e.g., oxalic acid, malonic acid,citric acid, fumaric acid, lactic acid, malic acid, succinic acid,tartaric acid, acetic acid, trifluoroacetic acid, gluconic acid,ascorbic acid, methylsulfonic acid, or benzylsulfonic acid), aninorganic base (e.g., sodium hydroxide, potassium hydroxide, calciumhydroxide, magnesium hydroxide, or ammonium hydroxide), an organic base(e.g., methylamine, diethylamine, triethylamine, triethanolamine,ethylenediamine, tris(hydroxymethyl)methylamine, guanidine, choline, orcinchonine), or an amino acid (e.g., lysine, arginine, or alanine) canbe used. Generally, nonaqueous media such as ether, ethyl acetate,ethanol, isopropanol, or acetonitrile are typical. Lists of suitablesalts are found in Remington's Pharmaceutical Sciences, 18th ed., MackPublishing Company, Easton, Pa., 1990, p. 1445, and Journal ofPharmaceutical Science, 66, 2-19 (1977). For example, they can be a saltof an alkali metal (e.g., sodium or potassium), alkaline earth metal(e.g., calcium), or ammonium.

The methods described herein comprise administering a compound offormula (I), formula (II), formula (III), or formula (IV), or apharmaceutically acceptable salt thereof in the form of a pharmaceuticalcomposition. In particular, a pharmaceutical composition will compriseat least one compound of formulas (I)-(IV), or a pharmaceuticallyacceptable salt thereof and a pharmaceutically acceptable carrier. Thepharmaceutically acceptable excipients described herein, for example,vehicles, adjuvants, carriers or diluents, are well-known to those whoare skilled in the art and are readily available to the public.Typically, the pharmaceutically acceptable carrier is one that ischemically inert to the active compounds and one that has no detrimentalside effects or toxicity under the conditions of use.

The pharmaceutical compositions can be administered as oral, sublingual,transdermal, subcutaneous, topical, absorption through epithelial ormucocutaneous linings, intravenous, intranasal, intraarterial,intramuscular, intratumoral, peritumoral, interperitoneal, intrathecal,rectal, vaginal, or aerosol formulations. In some aspects, thepharmaceutical composition is administered orally or intravenously.

In accordance with any of the embodiments, the compound of formula (I),formula (II), formula (III), or formula (IV), or a pharmaceuticallyacceptable salt thereof can be administered orally to a subject in needthereof. Formulations suitable for oral administration can consist of(a) liquid solutions, such as an effective amount of the compounddissolved in diluents, such as water, saline, or orange juice andinclude an additive, such as cyclodextrin (e.g., α-, β-, orγ-cyclodextrin, hydroxypropyl cyclodextrin) or polyethylene glycol(e.g., PEG400); (b) capsules, sachets, tablets, lozenges, and troches,each containing a predetermined amount of the active ingredient, assolids or granules; (c) powders; (d) suspensions in an appropriateliquid; and (e) suitable emulsions and gels. Liquid formulations mayinclude diluents, such as water and alcohols, for example, ethanol,benzyl alcohol, and the polyethylene alcohols, either with or withoutthe addition of a pharmaceutically acceptable surfactant, suspendingagent, or emulsifying agent. Capsule forms can be of the ordinary hard-or soft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers, such as lactose, sucrose, calciumphosphate, and cornstarch. Tablet forms can include one or more oflactose, sucrose, mannitol, corn starch, potato starch, alginic acid,microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicondioxide, croscarmellose sodium, talc, magnesium stearate, calciumstearate, zinc stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and pharmacologically compatiblecarriers. Lozenge forms can comprise the active ingredient in a flavor,usually sucrose and acacia or tragacanth, as well as pastillescomprising the active ingredient in an inert base, such as gelatin andglycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to the active ingredient, such carriers as areknown in the art.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The compound of formula (I), formula (II), formula (III), or formula(IV), or a pharmaceutically acceptable salt thereof can be administeredin a physiologically acceptable diluent in a pharmaceutical carrier,such as a sterile liquid or mixture of liquids, including water, saline,aqueous dextrose and related sugar solutions, an alcohol, such asethanol, isopropanol, or hexadecyl alcohol, glycols, such as propyleneglycol or polyethylene glycol, glycerol ketals, such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters. Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include (a)cationic detergents such as, for example, dimethyl dialkyl ammoniumhalides, and alkyl pyridinium halides, (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylene-polypropylene copolymers, (d) amphoteric detergents suchas, for example, alkyl-beta-aminopropionates, and 2-alkyl-imidazolinequaternary ammonium salts, and (3) mixtures thereof.

The parenteral formulations will typically contain from about 0.5 toabout 25% by weight of the inhibitors in solution. Suitablepreservatives and buffers can be used in such formulations. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5 to about15% by weight. Suitable surfactants include polyethylene sorbitan fattyacid esters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

The inhibitors may be made into injectable formulations. Therequirements for effective pharmaceutical carriers for injectablecompositions are well known to those of ordinary skill in the art. SeePharmaceutics and Pharmacy Practice, J. B. Lippincott Co., Philadelphia,Pa., Banker and Chalmers, eds., pages 238-250 (1982), and ASHP Handbookon Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986).

Topically applied compositions are generally in the form of liquids(e.g., mouthwash), creams, pastes, lotions and gels. Topicaladministration includes application to the oral mucosa, which includesthe oral cavity, oral epithelium, palate, gingival, and the nasalmucosa. In some embodiments, the composition contains at least oneactive component and a suitable vehicle or carrier. It may also containother components, such as an anti-irritant. The carrier can be a liquid,solid or semi-solid. In embodiments, the composition is an aqueoussolution, such as a mouthwash. Alternatively, the composition can be adispersion, emulsion, gel, lotion or cream vehicle for the variouscomponents. In one embodiment, the primary vehicle is water or abiocompatible solvent that is substantially neutral or that has beenrendered substantially neutral. The liquid vehicle can include othermaterials, such as buffers, alcohols, glycerin, and mineral oils withvarious emulsifiers or dispersing agents as known in the art to obtainthe desired pH, consistency and viscosity. It is possible that thecompositions can be produced as solids, such as powders or granules. Thesolids can be applied directly or dissolved in water or a biocompatiblesolvent prior to use to form a solution that is substantially neutral orthat has been rendered substantially neutral and that can then beapplied to the target site. In embodiments of the invention, the vehiclefor topical application to the skin can include water, bufferedsolutions, various alcohols, glycols such as glycerin, lipid materialssuch as fatty acids, mineral oils, phosphoglycerides, collagen, gelatinand silicone based materials.

The compound of formula (I), formula (II), formula (III), or formula(IV), or a pharmaceutically acceptable salt thereof, alone or incombination with other suitable components, can be made into aerosolformulations to be administered via inhalation. These aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen, and the like. They alsomay be formulated as pharmaceuticals for non-pressured preparations,such as in a nebulizer or an atomizer

The dose administered to the subject, particularly human and othermammals, in accordance with the present invention should be sufficientto affect the desired response. One skilled in the art will recognizethat dosage will depend upon a variety of factors, including the age,condition or disease state, predisposition to disease, genetic defect ordefects, and body weight of the mammal. The size of the dose will alsobe determined by the route, timing and frequency of administration aswell as the existence, nature, and extent of any adverse side-effectsthat might accompany the administration of a particular inhibitor andthe desired effect. It will be appreciated by one of skill in the artthat various conditions or disease states may require prolongedtreatment involving multiple administrations.

The inventive methods comprise administering a pharmaceuticallyeffective amount of a compound of formula (I), formula (II), formula(III), or formula (IV), or a pharmaceutically acceptable salt thereoffor the treatment of a disease. As used herein, the terms “treatment,”“treated,” “treating,” and the like refer to obtaining a desiredpharmacologic and/or physiologic effect. Preferably, the effect istherapeutic, i.e., the effect partially or completely cures a diseaseand/or adverse symptom attributable to the disease. A “pharmaceuticallyeffective amount” means an amount sufficient to show a meaningfulbenefit in an individual, e.g., promoting at least one aspect of tumorcell cytotoxicity (e.g., inhibition of growth, inhibiting survival of acancer cell, reducing proliferation, reducing size and/or mass of atumor (e.g., solid tumor)), or treatment, healing, prevention, delay ofonset, halting, or amelioration of other relevant medical condition(s)associated with a particular cancer. The meaningful benefit observed inthe patient can be to any suitable degree (e.g., at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, or 90% or more). In some aspects, one or moresymptoms of the cancer are prevented, reduced, halted, or eliminatedsubsequent to administration of a compound of formula (I), formula (II),formula (III), or formula (IV), including compounds of formula (Ia),(Ib), (Ic), (Id), (Ie), (If), (Ig), (IIa), (IIIa), and (IVa), or apharmaceutically acceptable salt thereof, thereby effectively treatingthe cancer to at least some degree.

Effective amounts can vary depending upon the biological effect desiredin the individual, condition to be treated, and/or the specificcharacteristics of the compound of formula (I), formula (II), formula(III), or formula (IV), including compounds of formula (Ia), (Ib), (Ic),(Id), (Ie), (If), (Ig), (IIa), (IIIa), and (IVa), or a pharmaceuticallyacceptable salt thereof, and the individual. In this respect, anysuitable dose of the compound of formula (I), formula (II), formula(III), or formula (IV), or a pharmaceutically acceptable salt thereofcan be administered to the patient (e.g., human), according to the typeof disease (e.g., cancer) to be treated. Various general considerationstaken into account in determining the “effective amount” are known tothose of skill in the art and are described, e.g., in Gilman et al.,Eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics,8th ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences,17th Ed., Mack Publishing Co., Easton, Pa., 1990, each of which isherein incorporated by reference. The dose of the compound of formula(I), formula (II), formula (III), or formula (IV), including compoundsof formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (IIa), (IIIa), and(IVa), or a pharmaceutically acceptable salt thereof desirably comprisesabout 0.1 mg per kilogram (kg) of the body weight of the mammal (mg/kg)to about 400 mg/kg (e.g., about 0.75 mg/kg, about 5 mg/kg, about 30mg/kg, about 75 mg/kg, about 100 mg/kg, about 200 mg/kg, or about 300mg/kg). In another embodiment, the dose of the compound formula (I),formula (II), formula (III), or formula (IV), or a pharmaceuticallyacceptable salt thereof, comprises about 0.5 mg/kg to about 300 mg/kg(e.g., about 0.75 mg/kg, about 5 mg/kg, about 50 mg/kg, about 100 mg/kg,or about 200 mg/kg), about 10 mg/kg to about 200 mg/kg (e.g., about 25mg/kg, about 75 mg/kg, or about 150 mg/kg), or about 50 mg/kg to about100 mg/kg (e.g., about 60 mg/kg, about 70 mg/kg, or about 90 mg/kg).

In an aspect of the invention, a compound of formula (I), a compound offormula (II), a compound of formula (III), or a compound of formula(IV), or a pharmaceutically acceptable salt thereof can inhibit theactivity of PRMT5 in a cell. Elevated activity of PRMT5 has beendescribed in the art for multiple disorders, including various types ofcancer (Wei et al., Proc. Natl. Acad. Sci., 110(33): 13516-13521 (2013);Lu et al., Cancer Res., 75(18): 3692-3695 (2015)). While not wishing tobe bound by any particular theory, it is believed that inhibition ofPRMT5 by a compound of formula (I), a compound of formula (II), acompound of formula (III), or a compound of formula (IV), or apharmaceutically acceptable salt thereof causes a decrease in theactivation of nuclear factor κB (NF-κB) leading to a decrease in tumorprogression.

As used herein the terms “elevated activity,” “increased activity” andthe like refers to any increase in the enzymatic activity of theprotein, any increase in the total amount of protein present, or anyincrease in the level of gene (e.g., mRNA, RNA, DNA) expression.Increased activity is measured against a control sample. For example,the mRNA level of PRMT5 is measured in a cancer cell and is compared tothe PRMT5 mRNA level in a non-cancer control cell of the same tissuetype. Methods to identify subjects or cells with increased activity of aprotein, increased level of a protein, or increased expression of a geneare routine in the art and include, for example, Western blotting,RT-PCR, PCR, quantitative PCR, ELISA, and enzyme assays. Additionally,the methods described herein can also be used by a person of ordinaryskill in the art to determine increased activity of a protein, increasedlevel of a protein or increased expression of a gene.

One aspect of the inventive method provides a method of treating cancerin a subject comprising administering a pharmaceutically effectiveamount of formula (I), formula (II), formula (III), or formula (IV),which includes the compounds of formulas (Ia), (Ib), (Ic), (Id), (Ie),(If), (Ig), (IIa), (IIIa), and (IVa), or a pharmaceutically acceptablesalt thereof, to the subject whereby the cancer is treated.

As used herein the term “subject” includes any living organism thatwould benefit from inhibition of PRMT5. In certain embodiments thesubject is a mammal. Mammals include, but are not limited to, the orderRodentia, such as mice, and the order Logomorpha, such as rabbits. Insome aspects, the mammals are from the order Carnivora, includingFelines (cats) and Canines (dogs), Artiodactyla, including Bovines(cows) and Swines (pigs) or of the order Perssodactyla, includingEquines (horses). In some aspects, the mammals are of the orderPrimates, Ceboids, or Simioids (monkeys) or of the order Anthropoids(humans and apes). In embodiments of the invention, the subject is ahuman.

The type of cancer is not particularly limited. In certain embodimentsthe cancer is characterized as a cancer that has increased activity ofPRMT5 compared to a non-cancer sample of the same tissue type. Theidentification of a cancer that has increased activity of PRMT5 can bereadily identified by a person of ordinary skill in the art usingroutine methods (e.g., Western blot, enzyme-linked immunosorbent assay(ELISA), or polymerase chain reaction (PCR)), including the methodsdescribed herein. In another embodiment the cancer is characterized as acancer that has increased activity of NF-κB compared to a non-cancersample of the same tissue type. The identification of a cancer that hasincreased activity of NF-κB can be readily identified by a person ofordinary skill in the art using routine methods (e.g., Western blot,ELISA, or PCR). In a further embodiment of the invention the cancer hasincreased activity of PRMT5 and increased activity of NF-κB.

Examples of cancers treatable with the inventive method include cancersof the head and neck, eye, skin, mouth, throat, esophagus, chest, bone,lung, colon, sigmoid, rectum, stomach, prostate, breast, ovaries,kidney, liver, pancreas, brain, intestine, heart, or adrenals. Moreparticularly, cancers include solid tumor, sarcoma, carcinomas,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, Kaposi's sarcoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, retinoblastoma, a blood-borne tumor, acutelymphoblastic leukemia, acute lymphoblastic B-cell leukemia, acutelymphoblastic T-cell leukemia, acute myeloblastic leukemia, acutepromyelocytic leukemia, acute monoblastic leukemia, acuteerythroleukemic leukemia, acute megakaryoblastic leukemia, acutemyelomonocytic leukemia, acutenonlymphocyctic leukemia, acuteundifferentiated leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia, hairy cell leukemia, or multiple myeloma. See,e.g., Harrison's Principles of Internal Medicine, Eugene Braunwald etal., Eds., pp. 491 762 (15th ed. 2001). In some aspects, the cancer is asolid tumor. In accordance with an embodiment, the cancer is selectedfrom gastrointestinal cancer, skin cancer, lung cancer, brain cancer,ovarian cancer, prostate cancer, lymphoma, melanoma, and breast cancer.In another embodiment, the cancer is pancreatic ductal adenocarcinoma(PDAC), colorectal cancer, pancreatic cancer, or liver cancer.

Another aspect of the inventive method provides a method of inhibitingthe activity of PRMT5 in a cell, comprising administering apharmaceutically effective amount of a compound of formula (I), formula(II), formula (III), or formula (IV), or a pharmaceutically acceptablesalt thereof to the cell, whereby the activity of PRMT5 is inhibited.Inhibition of PRMT5 includes any decrease in methyltransferase activitycompared to an untreated or control treated subject. Inhibition or PRMT5also includes any decrease in the protein level of PRMT5 in a subjectcompared to an untreated or control treated subject. Inhibition of PRMT5can be demonstrated, for example, by a decrease in NF-κB activity.

In certain embodiments the cell is treated in vitro or ex vivo. In otherembodiments the cell is in a subject.

The type of cell in which PRMT5 is inhibited is not particularlylimited. In certain embodiments, the cell is a cancer cell. The cancercell can be from any cancer type described in the foregoing. In certainembodiments the cancer cell is from PDAC, colorectal cancer, pancreaticcancer, or liver cancer.

PRMT5 activity has been shown to be increased in a variety of disease,and inhibition of PRMT5 has been described in the art as a viabletreatment of multiple diseases, including autoimmune diseases,inflammatory disease, metabolic disorders, neurological andneurodegenerative disorders, cardiovascular diseases, and blooddisorders (Kim et al., Mediators Inflamm., 2016; Published online doi:10.1155/2016/4028353; Stopa et al., Cell Mol. Life Sci., 2015; 72(11):2041-2059; Karkhanis et al., Trends Biochem Sci., 2011; 36(12): 633-641;Likhite et al., Science Signaling, 2015; 8(402) ra115; Wei et al., CellCycle, 2013; 13: 32-41; Kryukov et al. Science, 2016; epub10.1126/science.aad5214). Therefore, another aspect of the inventionprovides a method of treating a disease associated with increasedexpression of PRMT5 in a subject, comprising administering apharmaceutically effective amount of a compound of formula (I), formula(II), formula (III), or formula (IV), or a pharmaceutically acceptablesalt thereof to the subject, thereby treating the disease. Suitablediseases that can be treated include, for example, autoimmune diseases,inflammatory disease, metabolic disorders, neurological andneurodegenerative disorders, cardiovascular diseases, and blooddisorders.

Examples of autoimmune diseases treatable with the inventive methodinclude alopecia areata, autoimmune hemolytic anemia, autoimmunehepatitis, dermatomyositis, diabetes (type 1), juvenile idiopathicarthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome,idiopathic thrombocytopenic purpura, myasthenia gravis, myocarditis,multiple sclerosis, pemphigus/pemphigoid, pernicious anemia,polyarteritis nodosa, polymyositis, primary biliary cirrhosis,psoriasis, rheumatoid arthritis, scleroderma/systemic sclerosis,Sjögren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis,vitiligo, and granulomatosis with polyangiitis (Wegener's).

Examples of inflammatory diseases treatable with the inventive methodinclude ankylosing spondylitis, arthritis (e.g., osteoarthritis,rheumatoid arthritis (RA), psoriatic arthritis), gout, eczema,gastritis, splenitis, sinusitis, hepatitis, nephritis, psoriasis,vasculitis, atherosclerosis, sarcoidosis, pleurisy, asthma,atherosclerosis, Crohn's disease, colitis, dermatitis, diverticulitis,fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupuserythematous (SLE), nephritis, and ulcerative colitis.

Examples of metabolic disorders treatable with the inventive methodinclude diabetes (type 1 and type 2), phenylketonuira, and obesity.

Examples of neurological and neurodegenerative disorders treatable withthe inventive method include drug abuse (e.g., methamphetamineaddiction), Alzheimer's disease, amyotrophic lateral sclerosis, Angelmansyndrome, Asperger syndrome, autism, bipolar disorder, cerebralarteriosclerosis, Charcot-Marie-Tooth disease, chronic pain, Cushing'ssyndrome, Creutzfeldt-Jakob disease, dementia, Huntington's disease,inclusion body myositis, Parkinson's disease, and Reye syndrome.

Examples of cardiovascular disorders treatable with the inventive methodinclude coronary artery disease, angina, myocardial infarction, stroke,hypertensive heart disease, rheumatic heart disease, cardiomyopathy,arrhythmia, congenital heart disease, valvular heart disease, carditis,aortic aneurysms, peripheral artery disease, and venous thrombosis.

Examples of blood disorders treatable with the inventive method includeanemia, bleeding disorders, hemophilia, sickle cell anemia,hemoglobinopathy, β-thalassemia, and blood clots.

In certain embodiments of this method, a compound formula (I), acompound of formula (II), a compound of formula (III), or a compound offormula (IV), or a pharmaceutically acceptable salt thereof can beco-administered with an anti-cancer agent (e.g., a chemotherapeuticagent) and/or radiation therapy. In an aspect, the method comprisesadministering an amount of a compound or salt that is effective tosensitize the cancer cells to one or more therapeutic regimens (e.g.,chemotherapy or radiation therapy). The terms “co-administered” or“co-administration” refer to simultaneous or sequential administration.A compound may be administered before, concurrently with, or afteradministration of another compound.

One or more than one, e.g., two, three, or more anti-cancer agents canbe administered. In this regard, the present invention is directed apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a combination of a compound of formula (I), a compound offormula (II), a compound of formula (III), or a compound of formula(IV), or a pharmaceutically acceptable salt thereof and at least oneanti-cancer agent (e.g., chemotherapeutic agent).

Examples of anti-cancer agents include platinum compounds (e.g.,cisplatin, carboplatin, oxaliplatin), alkylating agents (e.g.,cyclophosphamide, ifosfamide, chlorambucil, nitrogen mustard, thiotepa,melphalan, busulfan, procarbazine, streptozocin, temozolomide,dacarbazine, bendamustine), antitumor antibiotics (e.g., daunorubicin,doxorubicin, idarubicin, epirubicin, mitoxantrone, bleomycin, mytomycinC, plicamycin, dactinomycin), taxanes (e.g., paclitaxel and docetaxel),antimetabolites (e.g., 5-fluorouracil, cytarabine, premetrexed,thioguanine, floxuridine, capecitabine, and methotrexate), nucleosideanalogues (e.g., fludarabine, clofarabine, cladribine, pentostatin,nelarabine), topoisomerase inhibitors (e.g., topotecan and irinotecan),hypomethylating agents (e.g., azacitidine and decitabine), proteasomeinhibitors (e.g., bortezomib), epipodophyllotoxins (e.g., etoposide andteniposide), DNA synthesis inhibitors (e.g., hydroxyurea), vincaalkaloids (e.g., vicristine, vindesine, vinorelbine, and vinblastine),tyrosine kinase inhibitors (e.g., imatinib, dasatinib, nilotinib,sorafenib, sunitinib), monoclonal antibodies (e.g., rituximab,cetuximab, panetumumab, tositumomab, trastuzumab, alemtuzumab,gemtuzumab ozogamicin, bevacizumab), nitrosoureas (e.g., carmustine,fotemustine, and lomustine), enzymes (e.g., L-Asparaginase), biologicalagents (e.g., interferons and interleukins), hexamethylmelamine,mitotane, angiogenesis inhibitors (e.g., thalidomide, lenalidomide),steroids (e.g., prednisone, dexamethasone, and prednisolone), hormonalagents (e.g., tamoxifen, raloxifene, leuprolide, bicaluatmide,granisetron, flutamide), aromatase inhibitors (e.g., letrozole andanastrozole), arsenic trioxide, tretinoin, nonselective cyclooxygenaseinhibitors (e.g., nonsteroidal anti-inflammatory agents, salicylates,aspirin, piroxicam, ibuprofen, indomethacin, naprosyn, diclofenac,tolmetin, ketoprofen, nabumetone, oxaprozin), selective cyclooxygenase-2(COX-2) inhibitors, or any combination thereof.

In other embodiments of this method, a compound formula (I), a compoundof formula (II), a compound of formula (III), or a compound of formula(IV), or a pharmaceutically acceptable salt thereof can beco-administered with an agent that treats a disease associated withincreased activity of PRMT5.

One or more than one, e.g., two, three, or more agents can beadministered. In this regard, the present invention is directed apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a combination of a compound of formula (I), a compound offormula (II), a compound of formula (III), or a compound of formula(IV), or a pharmaceutically acceptable salt thereof and at least oneother PRMT5 disease modifying agent.

Examples of autoimmune disease and inflammatory disease treating agentsinclude alkylating agents (e.g., cyclophosphamide, ifosfamide,chlorambucil, nitrogen mustard, thiotepa, melphalan, busulfan,procarbazine, streptozocin, temozolomide, dacarbazine, bendamustine),antimetabolites (e.g., methotrexate, azathioprine, mercaptopurine,flurouracil), cytotoxic antibiotics (e.g., daunorubicin, doxorubicin,idarubicin, epirubicin, mitoxantrone, bleomycin, mytomycin C,plicamycin, dactinomycin), polyclonal antibodies (e.g., atgam,thymoglobuline), monoclonal antibodies (e.g., muromonab-CD3,basiliximab, daclizumab, omalizumab), calcinurin and mTOR inhibitors(e.g., ciclosporin, tacrolumus, sirolimus, everolimus)), biologicalagents (e.g., interferons and interleukins), opioids, TNF bindingproteins (e.g., infliximab, etanercept, adalimumab, curcumin catechins),nonselective cyclooxygenase inhibitors (e.g., nonsteroidalanti-inflammatory agents, salicylates, aspirin, piroxicam, ibuprofen,indomethacin, naprosyn, diclofenac, tolmetin, ketoprofen, nabumetone,oxaprozin), selective cyclooxygenase-2 (COX-2) inhibitors,disease-modifying antirheumatic drugs (DMARDS) (e.g.,hydroxychloroquine, leflunomide, minocycline, anakinra, abatacept,azathioprine, tofacitinib), steroids (e.g., glucocorticoids, cortisol,prednisone, dexamethasone, and prednisolone), or any combinationthereof.

Examples of metabolic disorder treating agents include short-actinginsulin (e.g., humulin, novolin), rapid-acting insulin (e.g., insulinaspart, insulin glulisine, insulin lispro), intermediate acting insulin(e.g., insulin isophane), long-acting insulin (e.g., insulin degludec,insulin detemir, insulin glargine), amylinomimetic drugs (e.g.,pramlintide), alpha-glucosidase inhibitors (e.g., acarbose, miglitol),biguanides (e.g., metformin), dopamine agonist (e.g., bromocriptine),DPP-4 inhibitors (e.g., alogliptin, linagliptin, saxagliptin,sitagliptin), glucagon-like peptides (e.g., albiglutide, dulaglutide,exenatide, liraglutide), meglitinides (e.g., nateglinide, repaglinide),sodium glucose transporter 2 inhibitors (e.g., dapagliflozin,canagliflozin, empagliflozin), sulfonylureas (e.g., glimerpiride,gliclazide, glipizide, glyburide, chlorpropamide, tolazamide,tolbutamide), thiazolidinediones (e.g., rosiglitazone, pioglitazone), orany combination thereof.

Examples of neurological and neurodegenerative disorder treating agentsinclude opioid addiction (e.g., methadone, buprenorphine, naltrexone),nicotine addiction (e.g., bupropion, varenicline), alcohol addiction(e.g., naltrexone, acamprosate, disulfiram), cholinesterase inhibitors(e.g., donepezil, rivastigmine, galantamide), N-methyl-D-aspartate(NMDA) antagonists (e.g., memantine), dietary supplements (e.g.,caprylidene), glutamate level reducers (e.g., riluzole), L-DOPA drugs(e.g., levodopa, carbidopa, benserazide, tolcapone, entacapone),dopamine agonists (e.g., bromocriptine, pergolide, pramipexole,ropinirole, piribedil, cabergoline, apomorphine and lisuride), MAO-Binhibitors (e.g., safinamide, selegiline, rasagiline), or anycombination thereof.

Examples of cardiovascular disease and blood disorder treating agentsinclude antiplatelets (e.g., aspirin, clopidogrel, prasugrel,ticagrelor), angiotensin-converting enzyme inhibitors (e.g., benazepril,captpril, enalapril, fosinopril, lisinopril, perindopril, quinapril,ramipril, trandolapril), beta-blockers (e.g., acebutolol, atenolol,carvedilol, labetalol, metoprolol, nadolol, nebivolol, penbutolol,pindolol, propranolol), anticoagulants (e.g., warfarin, acenocoumarol,phenprocoumon, dabigatran, apixaban, edoxaban, rivaroxaban), clottingdrugs (e.g., Factor VIII, advate, tranexamic acid, desmopressin),hydroxyurea (e.g., droxia, hydrea), antibiotics (e.g., penicillin),statins (e.g., atorvastatin, fluvastatin, lovastatin, pravastatin,rosuvastatin, simvastatin, pitavastatin), vasodilators (e.g., nitricoxide, doxazosin, prazosin, terazosin, clonidine, hydralazine,minoxidil), or any combination thereof.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates the development of small-molecule PRMT5inhibitors in an embodiment of the invention.

In order to identify PRMT5 inhibitors, the AlphaLISA technique(PerkinElmer, Waltham, Mass.) was adapted to an assay that couldprecisely quantify PRMT5 dimethylation of its substrate in a 384-wellHTS platform (FIG. 1). The AlphaLISA high-throughput screen comprisesincubating a biotinylated substrate (e.g., histone H4), PRMT5, and amethyl donor (e.g., S-adenosyl-1-methionine (SAM)); PRMT5 dimethylatesthe biotinylated substrate and the dimethylated biotinylated substrateis recognized by Acceptor beads specific for the methylation site. Donorbeads comprising a tag (e.g., streptavadin) bind to the biotinylatedsubstrate, and interaction between the Acceptor beads and Donor beadsemits a signal (e.g., chemiluminescent signal), which can be detected.The methylation activity of PRMT5 is proportional to the intensity ofthe signal.

In one embodiment of the method used to identify the compounds disclosedherein, PRMT5 enzyme was purified with anti-Flag-M2 beads(Sigma-Aldrich, St. Louis, Mo.) from 293 cells overexpressing theFlag-PRMT5 protein (Wei et al., Proc Natl Acad Sci USA, 2013; 110:13516-13521). The enzyme prep was diluted at 1:10 in assay buffer (30 mMTris, pH 8.0, 1 mM DTT, 0.01% BSA, 0.01% Tween-20) before use. 100 μMS-adenosylmethionine (SAM) (New England Biolabs, Ipswich, Mass.) wasused as the methyl group donor and 30 nM unmethylated histone H4R3(Anaspec, Thornleigh, Australia) was used as a substrate. For screening,250 nL of 1 mM library compounds was added to each well with a finalcompound concentration at 12.5 μM in 1.25% of dimethylsulfoxide (DMSO).All these components were incubated at RT for 1 h. Acceptor beads anddonor beads were diluted 1:50 fold in 1× Epigenetics buffer(PerkinElmer, Waltham, Mass.) before use. Acceptor beads were then addedat a final concentration of 20 μg/ml to the reaction mixture and theplate was incubated at room temperature (RT) for 1 hour. Donor beadswere added at a final concentration of 20 μg/ml, and the plate wasincubated at RT for 30 min. The reaction was run in 384 well plates. Theplates were read using an ENVISION™ Reader (PerkinElmer, Waltham,Mass.).

A library of 10,000 small molecules was screened, and several potentialcompounds were identified to significantly inhibit PRMT5 methylationactivity. The PRMT5 inhibitory activity of the compounds from the screenwas confirmed using both AlphaLISA and an MTT [(3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide)] assay(PerkinElmer, Waltham, Mass.). For the MTT assay, cells were seeded at90% confluence in 96-well plates and titrated with different dosages ofcompound (IIa) for 4 days. Media was then removed and 100 μl of MTT(Sigma-Aldrich, St. Louis, Mo.) was added per well. Cells were incubatedfor 2 hours at 37° C. before adding 100 μl of DMSO to each well andquantified with the SYNERGY™ H1 Multi-Mode Reader (BioTek InstrumentsInc., Winooski, Vt.).

Amongst the hits, the leading compounds were a compound of formula (II),specifically compound (IIa), a compound of formula (III), specificallycompound (IIIa), and a compound of formula (IV), specifically compound(Iva). The IC₅₀ of compound (IIa) was 7.5 μM, the IC₅₀ of compound(IIIa) was ˜1.5 μM, and the IC₅₀ of compound (IVa) was 16.5 μM, asdetermined by the AlphaLISA (PerkinElmer, Waltham, Mass.) approach invitro (FIGS. 2A-2C). To determine whether compound (IIa) reduced p65methylation of NF-κB, Flag-p65 expressing cells were treated with 20 μMcompound (IIa) for 24 hours, Flag-p65 was then pulled down withanti-Flag-M2 beads and analyzed with Western blot by probing withanti-dimethylated arginine (FIG. 2B). Compared to the untreated control,treatment with compound (IIa) significantly inhibited PRMT5-mediated p65methylation. Additionally, compound (IIa) did not inhibit or had atleast a 10-fold higher IC₅₀ against other protein argininemethyltransferase family members when analyzed using the HotSpotradioisotope-based platform (Reaction Biology Corp, Malvern, Pa.)(Horiuchi et al., Assay Drug Dev. Technol., 2013; 11: 227-236).

These results indicate that a compound of formula (II), a compound offormula (III), and a compound of formula (IV) can selectively inhibitthe activity of PRMT5.

Example 2

This example describes the lead optimization of compound (IIa) toidentify small-molecule PRMT5 inhibitors with increased PRMT5 inhibitoryactivity compared to compound (IIa).

In order to identify small-molecule PRMT5 inhibitors with increasedefficacy compared to compound (IIa), lead optimization of compound (IIa)was performed. Briefly, the pyrozolo-pyrimidine core was maintained andperipheral groups were either added or modified. Compounds were testedto identify PRMT5 inhibitors and their corresponding IC₅₀ values usingan MTT assay. For the MTT assay cells (HT-29 or PANC1) were seeded at90% confluence in 96-well plates and titrated with different dosages ofcompound (IIa) for 4 days. Media was then removed and 100 μl of MTT(Sigma-Aldrich) was added per well. Cells were incubated for 2 hours at37° C. before adding 100 μl of DMSO to each well and quantified with theSynergy H1 Multi-Mode Reader (BioTek Instruments Inc., Winooski, Vt.).

Amongst the compounds, the IC₅₀ of compound (Ia) was 2 μM in HT-29 andPANC1 cells, the IC₅₀ of compound (If) was 7 μM in HT-29 and 8 μM inPANC1 cells, the IC₅₀ of compound (Ig) was 7 μM in HT-29 and 11 μM inPANC1 cells, and the IC₅₀ of compound (Ib) was 11 μM in PANC1 cells(FIGS. 3A and 3B).

These results indicate that compounds of formula (I) can selectivelyinhibit the activity of PRMT5.

Example 3

This example demonstrates that PRMT5 expression is increased in multipletypes of cancer.

The expression of PRMT5 was examined in pancreatic ductal adenocarcinomacells at multiple stages of disease progression and in multiplecolorectal cancer cell lines. Pancreatic control (HPNE) and cancer celllines (PANC1, MiaPaCa2 and AsPC1) were grown in Dulbecco's ModifiedEagle Medium (DMEM) (GE Healthcare, Little Chalfont, UK), supplementedwith 1% of penicillin/streptomycin, 10% fetal bovine serum (FBS). Coloncontrol (FHC) and cancer (HT29, HCT116, and DLD1) cell lines weremaintained in RPMI 1640 Medium (Roswell Park Memorial Institute Medium)(GE Healthcare, Little Chalfont, UK), containing 1%penicillin/streptomycin and 10% FBS, while FHC cells were cultured underthe same condition with further addition of 25 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 10 ng/ml choleratoxin, 0.005 mg/ml insulin, 0.005 mg/ml transferring, and 100 ng/mlhydrocortisone. All cell lines were cultured at 37° C. under 5% CO₂.

The expression of PRMT5 in the cell lines was determined by Westernblotting using an anti-PRMT5 specific antibody. As shown in FIGS. 4A and4C, expression of PRMT5 was markedly increased in each of the cell linestested as compared to the corresponding control cell lines.

The expression of PRMT5 was also analyzed in tissue microarray samplesusing immunohistochemistry. Pancreatic and colon cancer tissuemicroarrays with matched normal adjacent controls were acquired fromOriGene Technologies (Rockville, Md.). The tissue microarrays wereblocked using protein-blocking solution (Dako Corporation, Carpinteria,Calif.) for 30 min. All subsequent staining steps were performed usingthe Dako FLEX SYSTEM and an automated immunostainer. Incubations werecarried out at room temperature and tris buffered saline containing0.05% TWEEN™ 20, pH 7.4 (Dako Corporation, Carpinteria, Calif.) was usedfor all the washes and diluents. Anti-PRMT5 primary antibody (Abcam,Cambridge, UK) was used to detect PRMT5 localization. Horseradishperoxidase conjugated to a secondary antibody was then used, followed byaddition of the chromogen, which formed a brown precipitate at thebinding site of secondary antibody. Imaging was done using an Aperiowhole slide digital imaging system (Leica Biosystems, Buffalo Grove,Ill.). The system imaged all slides at 20× magnification.

The expression of PRMT5 was significantly higher in various stages ofPDAC (FIG. 4B), particularly in the metastatic stage, as compared to thenormal PDAC adjacent pancreatic tissue. Similarly, PRMT5 had much higherexpression in samples ranging from inflammation, poylp, to themetastatic stage of CRC as compared to CRC adjacent normal colon tissue(FIG. 4D).

Therefore, these results indicate that the expression of PRMT5 isincreased compared to controls in multiple cancer cell types.

Example 4

This example demonstrates that PRMT5 promotes cell proliferation,anchorage-independent growth, and cell migration is cells with increasedPRMT5 expression.

The effect of PRMT5 on various characteristics of cancer cells,including cell proliferation, anchorage-independent growth, as well as,cell migration was examined. First, stable cell lines were generated,with either PRMT5 overexpression or shRNA knockdown of PRMT5 in PANC1(PDAC) and HT29 (CRC) cell lines. Western blotting was carried out toverify the stable overexpression or knockdown of PRMT5 in these celllines. As shown in FIG. 5A, the stable cell lines showed desiredexpression level of PRMT5, as expected. Using the above cell models, theeffect of PRMT5 on cell growth was determined. As shown in FIGS. 5C and5D, overexpression of PRMT5 led to a significant increase in the cellgrowth, while knockdown of PRMT5 showed opposite effect in both PDAC andHT29 derived stable lines, strongly suggesting that PRMT5 is a promoterfor cell proliferation.

The ability to form colonies suspended in soft agar is a characteristicof cancer cells. By using the stable cell lines established above, ananchorage-independent assay in soft agar was conducted. 2.5% and 1.25%agar were used to prepare the bottom and top layers of the soft agar,respectively. The bottom agar was added to each well of a 6-well plate.2×10⁵ cells for each cell line were then added and mixed into top agarsolution and layered on top of the bottom layer. The plates wereincubated for 10-20 days at 37° C. and 5% CO₂. Images were capturedusing a Canon EOS Rebel T3i Digital SLR camera (Canon, Woodridge, Ill.)and quantification of colony size and number was performed using ImageJ.PRMT5 overexpression led to a significant increase in not only colonysize but also colony number, while shPRMT5 knockdown showed the oppositeeffect in both PANC1 (FIG. 5D) and HT29 (FIG. 5E) cells. This resultconfirmed that PRMT5 played a critical role in promotinganchorage-independent cell growth in cells with increased PRMT5expression.

Another important common feature for cancers is the ability formetastasis. Cancer cells often have a stronger migration ability thannormal cells, which is critical for tumor invasion and metastases (Shaw,Methods Mol Biol, 2005; 294: 97-105). Boyden chamber assays wereconducted to determine the effect of PRMT5 on the invasive nature ofcells with increased PRMT5 activity. 8 μm pore size cell culture inserts(Corning Inc., Corning, N.Y.) were placed in a 24 well plate. Eachchamber was coated with gelatin on the side facing the lower chamber.2×10⁵ cells were suspended in serum-starved media in the upper chamberof the well. Corresponding serum rich media was added to the lowerchamber. Migrated cells were fixed using 4% formaldehyde followed bycrystal violet staining and counting using a microscope at 20×magnification. The images were captured using a Canon EOS Rebel T3iDigital SLR camera (Canon, Woodridge, Ill.). As observed in FIG. 5F,overexpression of PRMT5 resulted in an increase in the number of cellsthat have been migrated, whereas shPRMT5 knockdown reduced this abilityas compared to the control cells. Similar effects in CRC cell lines, asshown in FIG. 5G, were also observed. These results pointed out theimportant role that PRMT5 plays in cell migration.

Taken together, these data show that increased expression of PRMT5 canlead to tumor activity.

Example 5

This example demonstrates the in vitro and in vivo efficacies of acompound of formula (I).

The PDAC and CRC cell lines described above were treated with increasingconcentrations of compound (Ia) and quantified for cell viability usingthe MTT assay described above. As shown in FIGS. 6A and 6B andsummarized in FIG. 6C, compound (Ia) had an IC₅₀ of 4.3 μM in PDAC cells(PANC1), and an IC₅₀ of 2.2 μM in CRC cells (HT29).

Experiments were also conducted to determine whether compound (Ia)exhibits tumor inhibition effects in mice xenograft models of cancerwith increased PRMT5 activity. NSG (NOD scid gamma) mice were obtainedfrom the In Vivo Therapeutics Core at Indiana University School ofMedicine. After acclimation for 7 days, NSG mice (6-8 weeks old) werexenografted with mycoplasma-free PANC1 or HT29 cells subcutaneously(1×10⁷ PANC1 or 3×10⁶ HT29 cells used per mouse in 0.2 ml of a 1:1 mixof phosphate-buffered saline and MATRIGEL™) (BD Biosciences, San Jose,Calif.). Mice were randomized when tumor volumes reached about 100 mm³.Mice were treated with either vehicle control or 20 mg/kg of compound(Ia) (drug stock dissolved in 1:1 CREMOPHOR™:ethanol solution)intraperitoneally 3 times per week. Tumor volumes and body weights weremeasured twice a week. The study was performed in accordance with theguidelines and standards of the Institutional Animal Care and UseCommittee (IACUC). As shown in FIGS. 7A and 7B, injection of compound(Ia) did not visibly affect the body weight of the implanted mice,however, it led to significant tumor inhibition effect in both PANC1(FIG. 7C) and HT29 (FIG. 7D) xenografted mice, demonstrating the stronganti-tumor efficacy of compound (Ia) against PRMT5 expressing cancers.

Example 6

This example demonstrates the in vitro and in vivo efficacies of acompound of formula (II).

The PDAC and CRC cell lines described above were treated with increasingconcentrations of compound (IIa) or the commercially available PRMT5inhibitor EPZ015666

and quantified for cell viability using the MTT assay described above.As shown in FIGS. 8A to 8F and summarized in FIG. 8G, compound (IIa) hada range of IC₅₀ at 2-4 μM in PDAC cells (PANC1, MiaPaCa2 and AsPC1), anda range of IC₅₀ at 10-11 μM in CRC cells (HT29, HCT116 and DLD1). A softagar experiment was also performed to determine the effect of compound(IIa) on colony growth. As indicated in FIG. 8H, treatment with compound(IIa) strongly inhibited colony formation of both PDAC and CRC cells.

The commercially available PRMT5 inhibitor, EPZ015666, was much lesseffective as compared to compound (IIa), with a range of IC₅₀ at 50-95μM for PDAC cells, and a range of IC₅₀ at 180-195 μM for CRC cells(FIGS. 9A-9F).

Experiments were also conducted to determine whether compound (IIa)exhibits tumor inhibition effects in mice xenograft models of cancerwith increased PRMT5 activity. NSG (NOD scid gamma) mice were obtainedfrom the In Vivo Therapeutics Core at Indiana University School ofMedicine. After acclimation for 7 days, NSG mice (6-8 weeks old) werexenografted with mycoplasma-free PANC1 or HT29 cells subcutaneously(1×10⁷ PANC1 or 3×10⁶ HT29 cells used per mouse in 0.2 ml of a 1:1 mixof phosphate-buffered saline and MATRIGEL™) (BD Biosciences, San Jose,Calif.). Mice were randomized when tumor volumes reached about 100 mm³.Mice were treated with either vehicle control or 20 mg/kg of compound(IIa) (drug stock dissolved in 1:1 CREMOPHOR™:ethanol solution)intraperitoneally 3 times per week. Tumor volumes and body weights weremeasured twice a week. The study was performed in accordance with theguidelines and standards of the Institutional Animal Care and UseCommittee (IACUC). As shown in FIGS. 10A and 10B, injection of compound(IIa) did not visibly affect the body weight of the implanted mice,however, it led to significant tumor inhibition effect in both PANC1(FIG. 10C) and HT29 (FIG. 10D) xenografted mice, demonstrating thestrong anti-tumor efficacy of compound (IIa) against PRMT5 expressingcancers.

Example 6

This example demonstrates that a PRMT5 inhibitor of formula (II)inhibits NF-κB activation and its target gene expression in cells withincreased expression of PRMT5.

Previously, it was discovered that PRMT5 activates NF-κB throughmethylation of its p65 subunit in HEK293 cells (Wei et al., Proc NatlAcad Sci USA, 2013; 110: 13516-13521; Wei et al., Cell Cycle, 2013; 13:32-41). As shown in FIGS. 11A and 11B, overexpresion of PRMT5 enhancesNF-κB activity, while PRMT5 knockdown causes the opposite effect in bothPANC1 (FIG. 11A) and HT-29 (FIG. 11B) cells. To determine if compound(IIa) decreases NF-κB activity, cells transfected with a NF-κBluciferase construct were treated with increasing concentrations ofcompound (IIa) or the control compound EPZ015666. As shown in FIGS. 11Cand 11D, treatment with increasing concentrations of compound (IIa)resulted in a corresponding decrease in NF-κB activation in PANC1 (FIG.11C) as well as HT29 cells (FIG. 11D). In great contrast, a much higherconcentration of EPZ015666 was required to observe the same effect(FIGS. 11C and 11D). These results demonstrate the high efficacy ofcompound (IIa) to decrease the NF-κB activation in PDAC as well as CRCcells.

The effect of overexpression of PRMT5 on downstream targets of NF-κB wasalso analyzed. As shown in FIGS. 11E-11H, overexpression of PRMT5significantly enhanced IL-1β-triggered typical NF-κB target genesexpression, TNFα and IL8, while shPRMT5 exhibited the opposite effect,in both PANC1 (FIGS. 11E and 11F) and HT29 cells (FIGS. 11G-11H).

Furthermore, experiments were performed to determine if PRMT5 inhibitionby compound (IIa) could decrease the expression of NF-κB target genes(e.g., pro-inflammatory cytokines TNFα and IL-8). Briefly, HT29 andPANC1 cells were treated with or without compound (IIa), and target geneexpression was analyzed by quantitative PCR. Treatment with compound(IIa) led to a significant decrease in the expression of TNFα and IL8 inboth PANC1 (FIGS. 11I-11J) and HT29 cells (FIGS. 11K-11L), thusindicating that compound (IIa) decreased the PRMT5-mediatedNF-κB-dependent gene activation. Overall, compound (IIa) showedsignificant efficacy in targeted reduction of NF-κB activation as wellas its downstream gene activation in both PDAC and CRC cells.

Therefore, taken together, these data show that the PRMT5 inhibitorsdisclosed herein can reduce that activity of NF-κB and subsequentlyreduce the expression of NF-κB target genes. Additionally, these datademonstrate that compound (IIa) can be used to treat inflammatory andautoimmune diseases via the inhibition of TNF and IL-8.

Example 7

This example demonstrates the efficacy of a compound of formula (III)and a compound of formula (IV).

The PDAC and CRC cell lines described above were treated with increasingconcentrations of compound (IIIa) or compound (IVa) and quantified forcell viability using the MTT assay described above. As shown in FIGS.12A to 12L and summarized in FIG. 12M, compound (IIIa) had a range ofIC₅₀ at 8.5-12.5 μM in PDAC cells (PANC1, MiaPaCa2 and AsPC1), and arange of IC₅₀ at 5-11 μM in CRC cells (HT29, HCT116 and DLD1), andcompound (IVa) had a range of IC₅₀ at 22-51 μM in PDAC cells (PANC1,MiaPaCa2 and AsPC1), and a range of IC₅₀ at 35-50 μM in CRC cells (HT29,HCT116 and DLD1).

These results demonstrate the strong anti-tumor efficacy of a compoundof formula (III) and a compound of formula (IV) against PRMT5 expressingcancers.

Example 8

This example demonstrates that PRMT5 activity is increased inneurological and neurodegenerative disorders.

The expression of PRMT5 was analyzed by Western blot is samples frommodels of Alzheimer's disease. Briefly, samples were taken fromnon-transgenic B6 mice (6M) (control), hTau (humanized tau mice) (3M),hTau (6M), htau;Trem2−/− mice (6M), hTau (18M), and APPPS1 amyloid mice.Cells overexpresssing PRMT5 were also used as a control. From thesedata, APPPS1 mice showed a significant increase in PRMT5 proteinexpression compared to age-matched B6 control mice (FIGS. 13A and 13B).Additionally, PRMT5 expression is increased along the aging of anotherAlzheimer's disease mouse model-hTau expression model and hTau/Trem2−/−mouse model.

Taken together, these data show that PRMT5 activity is increased inneurological and neurodegenerative disorders, and indicate thatinhibition of PRMT5 with a compound of formula (I), formula (II),formula (III), or formula (IV), or a pharmaceutically acceptable saltthereof can be an effective treatment.

Example 9

This example demonstrates that a compound of formula (II) can be used totreat a neurological disorder.

Methamphetamine induces inflammation and activation of microglia throughNFκB. Increased NFκB promotes inflammation and excitotoxicity areassociated with neuronal damage and decrease in neurotransmitter (NT)levels of dopamine and serotonin. Thus inhibition of NF-κB via compound(IIa) mediated inhibition of its activator, PRMT5 could reduceneurotoxicity observed during post-meth administration and restore NTlevels. This data suggests that compound (IIa) may be used to treatmethamphetamine abuse.

Rats were given methamphetamine treatment 7.5 mg/kg, 4 injections spacedout at 2 hours each. Then, 4 injections of 1 mg/kg of compound (IIa) orvehicle control were given at 12 hours, 24 hours, 36 hours, and 48 hoursafter the last methamphetamine injection. Rats were sacrificed 24 hoursafter the last injection and neurotransmitter levels were quantified inthe striatum using high performance liquid chromatography (HPLC). In thegroup administered the compound of formula (II), an increase in thelevels of NT levels were observed in the striatum region of the rats,thereby suggesting a neuroprotective effect (FIGS. 14A and 14B).

These data show that treatment with a compound of formula (II) can beused for the treatment of neurological disorders. Moreover, these dataindicate that compounds of formula (I), formula (III), or formula (IV),or a pharmaceutically acceptable salt thereof may be effective for thetreatment of neurological disorders.

Example 10

This example demonstrates that PRMT5 is highly expressed in the heart.

The expression level of PRMT5 was analyzed in multiple tissues using theTiGER database. The expression profile PRMT5 in humans shows asignificant expression of PRMT5 in heart tissue (FIG. 15).

These data indicate that inhibition of PRMT5 with a compound of formula(I), formula (II), formula (III), or formula (IV), or a pharmaceuticallyacceptable salt thereof may be an effective treatment for cardiovasculardisease.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A compound of formula (II):

wherein R⁵ is halo, aryl, substituted aryl, heterocycloalkyl, or heteroaryl; R⁶ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, C₁-C₈ alkoxy, C₃-C₆ cycloalkyloxy, aryloxy, halo, C₁-C₈ haloalkoxy, C₁-C₈ haloalkyl, haloaryl, haloaryloxy, —CN, —NO₂, —(CH₂)_(n)C(O)R⁹, —(CH₂)_(n)CO₂R⁹, —(CH₂)_(n)C(O)NR⁹R¹⁰, —(CH₂)_(n)NR⁹C(O)R¹⁰, —(CH₂)_(n)NR⁹R¹⁰, —NR¹¹(CH₂)_(n)NR⁹R¹⁰; or two R⁶ moieties and the phenyl group to which they are attached form a naphthyl group that is optionally substituted; R⁷ is hydroxy, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkylalkyl, C₁-C₈ alkoxy, C₃-C₆ cycloalkyloxy, heterocycloalkyl, aryl, arylalkyl, heteroaryl, aryloxy, halo, C₁-C₈ haloalkyl, C₁-C₈ haloalkoxy, haloaryl, haloaryloxy, —CN, —NO₂, —C(O)R⁹, —CO₂R⁹, —C(O)NR⁹R¹⁰, —NR⁹C(O)R¹⁰, —(CH₂)_(n)NR⁹R¹⁰, —(CH₂)_(n)SO₂NR⁹R¹⁰, —(CH₂)_(n)SO₂R⁹, aryl; or two R⁷ moieties and the phenyl group to which they are attached form a naphthyl group that is optionally substituted; R⁸ is H, hydroxy, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₁-C₈ haloalkyl, —CN, —NO₂, —(CH₂)_(n)NR⁹R¹⁰, heterocycloalkyl, aryl, or heteroaryl; X is —(CH₂)_(o)CR⁹R¹⁰—, —CR⁹R¹⁰(CH₂)_(o)—, —(CH₂)_(o)NR⁹—, —NR⁹(CH₂)_(o)—, —(CH₂)_(o)O—, or —O(CH₂)_(o)—; R⁹, R¹⁰, and R¹¹ are independently selected from the group consisting of H and C₁-C₈ alkyl; m, n, and o are each an integer independently selected from the range of 0-5; or a pharmaceutically acceptable salt thereof.
 2. The compound of claim 1, wherein R⁵ is halo, aryl, substituted aryl, heterocycloalkyl, or heteroaryl; R⁶ is C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkylalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, C₁-C₈ alkoxy, C₃-C₆ cycloalkyloxy, aryloxy, halo, C₁-C₈ haloalkoxy, C₁-C₈ haloalkyl, haloaryl, haloaryloxy, —CN, —NO₂, —(CH₂)_(n)C(O)R⁹, —(CH₂)_(n)CO₂R⁹, —(CH₂)_(n)C(O)NR⁹R¹⁰, —(CH₂)_(n)NR⁹C(O)R¹⁰, —(CH₂)_(n)NR⁹R¹⁰, or —NR¹¹(CH₂)_(n)NR⁹R¹⁰; R⁷ is hydroxy, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₃-C₆ cycloalkylalkyl, C₁-C₈ alkoxy, C₃-C₆ cycloalkyloxy, aryloxy, halo, C₁-C₈ haloalkyl, C₁-C₈ haloalkoxy, haloaryl, haloaryloxy, —CN, —NO₂, —C(O)R⁹, —CO₂R⁹, —C(O)NR⁹R¹⁰, —NR⁹C(O)R¹⁰, —(CH₂)_(n)NR⁹R¹⁰, —(CH₂)_(n)SO₂NR⁹R¹⁰, or —(CH₂)_(n)SO₂R⁹; R⁸ is H, hydroxy, C₁-C₈ alkyl, C₂-C₈ alkenyl, C₃-C₆ cycloalkyl, C₁-C₈ haloalkyl, —CN, —NO₂, —(CH₂)_(n)NR⁹R¹⁰, heterocycloalkyl, aryl, or heteroaryl; X is —(CH₂)_(o)CR⁹R¹⁰—, —CR⁹R¹⁰(CH₂)_(o)—, —(CH₂)_(o)NR⁹—, —NR⁹(CH₂)_(o)—, —(CH₂)_(o)O—, or —O(CH₂)_(o)—; R⁹, R¹⁰, and R¹¹ are independently selected from the group consisting of H and C₁-C₈ alkyl; m, n, and o are each an integer independently selected from the range of 0-5.
 3. The compound of claim 2, wherein R⁶ is —NR¹¹(CH₂)_(o)NR⁹R¹⁰.
 4. The compound of claim 2, wherein R⁷ is C₁-C₈ alkyl, halo, or C₁-C₈ haloalkyl.
 5. The compound of claim 2, wherein R⁸ is H.
 6. The compound of claim 2, wherein X is —(CH₂)_(o)NR⁵— or —NR⁵(CH₂)_(o)—.
 7. The compound of claim 1, wherein m is
 1. 8. The compound of claim 6, wherein o is 1 or
 2. 9. The compound of claim 1, wherein R⁵ is piperazinyl, pyrrolyl, pyranyl, piperidyl, morpholinyl, phenyl, substituted phenyl, and pyrrolidinyl; R⁶ is —NR¹¹(CH₂)_(n)NR⁹R¹⁰; R⁷ is H, C₁-C₈ alkyl, halo, or C₁-C₈ haloalkyl; R⁸ is H; X is —NR⁹—, —(CH₂)_(o)CR⁹R¹⁰—, —CR⁹R¹⁰(CH₂)_(o)—, or —(CH₂)_(o)NR⁹—; R⁹, R¹⁰, and R¹¹ are independently selected from H or C₁-C₈ alkyl; m is 1; and o is 1 or
 2. 10. The compound of claim 9 wherein R⁵ is a 3,4-di-C₁-C₈ alkylphenyl, optionally wherein the 3,4-di-C₁-C₈ alkylphenyl is 3,4-dimethylphenyl.
 11. The compound of claim 1, wherein the compound is


12. A pharmaceutical composition comprising a compound of claim 1 or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
 13. A method of treating a cancer in a subject, comprising administering the pharmaceutical composition of claim 12 or a pharmaceutically acceptable salt thereof to the subject, whereby the cancer is treated.
 14. The method of claim 13, wherein the cancer has (i) increased activity of protein arginine methyltransferase 5 (PRMT5) compared to a non-cancer cell from the subject; and/or (ii) the cancer has a constitutive activation of nuclear factor κB (NF-κB); and/or (iii) the cancer is a gastrointestinal cancer, skin cancer, lung cancer, brain cancer, ovarian cancer, prostate cancer, lymphoma, melanoma, or breast cancer; and wherein the gastrointestinal cancer is optionally selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), colorectal cancer, pancreatic cancer, and liver cancer.
 15. The method of claim 14, wherein the compound or pharmaceutically acceptable salt thereof is administered in a composition comprising the compound or a salt thereof and a pharmaceutically acceptable carrier.
 16. A method of inhibiting the activity of PRMT5 in a cell, comprising administering a compound of claim 1, or a pharmaceutically acceptable salt thereof, to the cell, whereby the activity of PRMT5 is inhibited.
 17. The method of claim 16, wherein (i) the cell is a cancer cell; and/or (ii) the cancer cell is optionally selected from a gastrointestinal cancer, skin cancer, lung cancer, brain cancer, ovarian cancer, prostate cancer, lymphoma, melanoma, or breast cancer; and wherein the gastrointestinal cancer is optionally selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), colorectal cancer, pancreatic cancer, and liver cancer.
 18. The method of claim 16 wherein said cell is present in a subject having a disease associated with increased activity of PRMT5, said method comprising administering a pharmaceutically effective amount of a compound of claim 1 or a pharmaceutically acceptable salt thereof to the subject.
 19. The method of claim 18, wherein the disease associated with increased activity of PRMT5 is a cancer, an autoimmune disease, an inflammatory disease, a metabolic disorder, a neurological disorder, a cardiovascular disorder, or a blood disorder. 